of research achievements during FY 2007.? ? A. Endocannabinoid signaling is critical for habit formation? Extended training can induce a shift in behavioral control from voluntary goal-directed actions, which are governed by action-outcome contingencies and sensitive to the value of the outcome, to habits which are insensitive to the outcome value. Previous studies in rats have shown that interval schedules of reinforcement favor habit formation while ratio schedules favor goal-directed behavior. However, the molecular mechanisms underlying habit formation are not well understood. Endocannabinoids, which can function as retrograde messengers acting through presynaptic CB1 receptors, are highly expressed in the dorsolateral striatum, a key region involved in habit formation. Using a reversible devaluation paradigm, we confirmed that in mice random interval schedules also favor habit formation compared with random ratio schedules. We also found that training with interval schedules resulted in a preference for exploration of a novel lever, whereas training with ratio schedules resulted in more exploitation of the reinforced lever. Furthermore, mice carrying either a heterozygous or a homozygous null mutation of the cannabinoid receptor type I (CB1) showed reduced habit formation, and enhanced exploitation. The impaired habit formation in CB1 mutant mice cannot be attributed to chronic developmental or behavioral abnormalities, because pharmacological blockade of CB1 receptors specifically during training also impairs habit formation. Taken together our data suggest that endocannabinoid signaling is critical for habit formation.? ? B. Subregion specific striatal changes during skill learning.? In the process of optimizing the action or making it more efficient, there is an initial stage of rapid increment in performance which is followed by slow increment as performance reaches asymptotic levels. The objective of our study was to investigate the changes in dorsal striatum during the different stages of skill learning; and to via which resolve the mechanism these changes occur. Mice were trained on an accelerating rotarod for either 1 day (one session of 10 trials, lightly trained, LT), or for 8 sessions over 14 days, well after their performance plateaued. After training, animals were left in their home cage undisturbed for at least 48 hours, after which coronal slices 350 um thick were cut, and field potential recordings of population spike in striatum were performed. We measured the input-output function of stimulation intensity vs. population spike amplitudes in the dorso-medial striatum (DMS, or associative striatum) and the dorso-lateral striatum (DLS, or sensorimotor striatum) in naive and yoked mice, LT and OT mice. We observed that in nave animals the input-output curves were higher in DMS than DLS. However, in LT animals both DMS and DLS showed higher input-output curves, and in OT animals DLS I-O curves were significantly higher than in DMS. Because changes in population spike amplitude could represent not only changes in synaptic strength but also other changes, like changes in excitability, we examined the capacity to induce long-term depression (LTD) in DMS and DLS of the experimental and control groups. Two trains of high frequency stimulation (HFS) were given at the half-maximal amplitude as determined by the I-O curve. The HFS was repeated every 30 minutes the field potential response was saturated. We found that the DLS synapses in the OT animals are stronger than in nave animals because LTD could be saturated further, and they took further trains to induce saturation. On the contrary, the DMS synapses were weaker in OT than in nave animals, and took fewer trains to saturate. In LT animals, the changes observed in the input-output function did not correspond to changes in synaptic strength, suggesting the possibility that they were cause by other mechanisms, like changes in excitability. These data indicate that extensive training in a skill leads to subregion specific plastic changes in striatum, with potentiation of the sensorimotor striatum in detriment of the associative striatum.? ? C. In vivo detection of changes in gene expression in the brain of awake behaving mice using fiber optics (In collaboration with Dr. Steve Vogel).? Most techniques used to detect changes in gene expression in behaving animals, such as in situ hybridization, western blot and immunocytochemistry, require posthoc processing of tissues obtained from the animals. To monitor real time local gene expression in awake and freely moving mice at different stages of behavioral training, we have designed a fiber optics system, which uses the principles of Time Correlated Single Photon Counting (TCSPC) to allow acquisition of emission spectra as well as lifetimes. The system is based on delivering pulse laser light of 473 nm through a single mode optical fiber to excite a sample, while a multimode fiber is used to collect the excitation emission. By using this system, we have been able to accurately differentiate GFP and Venus signals in solutions containing these two proteins at different ratios as well as calculate taus for GFP averaging 2.95 +/- 0.4 ns. This average correlates with the published taus of between 2.4 and 2.6 ns. We were also able to use this system to detect GFP expression in subcortical brain structures, like the striatum of GAD-GFP mice. We are in the process of applying this system to monitor the expression of immediate early genes in the dorsal striatum during learning. By monitoring the relative ratios of GFP to Venus in mice expressing both GFP as a reporter of activty-dependent changes in gene expression, and Venus as a constitutively active marker marker of cells, we can investigate changes in the expression of immediate early genes during learning.
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